Arch Microbiol (2012) 194:759–767

DOI 10.1007/s00203-012-0811-4

ORIGINAL PAPER

Role of altered rpoB alleles in Bacillus subtilis sporulation and spore resistance to heat, hydrogen peroxide, formaldehyde, and glutaraldehyde

Ralf Moeller · Ignacija VlaniT · Günther Reitz · Wayne L. Nicholson

Received: 6 December 2011 / Revised: 21 March 2012 / Accepted: 23 March 2012 / Published online: 8 April 2012© Springer-Verlag 2012

Abstract Mutations in the RNA polymerase �-subunitgene rpoB causing resistance to rifampicin (RifR) in Bacil-lus subtilis were previously shown to lead to alterations inthe expression of a number of global phenotypes known tobe under transcriptional control. To better understand theinXuence of rpoB mutations on sporulation and spore resis-tance to heat and chemicals, cells and spores of the wild-type and twelve distinct congenic RifR mutant strains ofB. subtilis were tested. DiVerent levels of glucose cataboliterepression during sporulation and spore resistance to heatand chemicals were observed in the RifR mutants, indicatingthe important role played by the RNA polymerase �-subunit,not only in the catalytic aspect of transcription, but also inthe initiation of sporulation and in the spore resistanceproperties of B. subtilis.

Keywords Bacillus subtilis · Sporulation · Spore resistance · Heat · Chemicals

Introduction

Bacillus subtilis is considered a ubiquitous microorganismthat can be isolated from almost every niche in nature(Nicholson et al. 2000, 2002; Horneck et al. 2010). Dor-mant spores exhibit astounding longevity up to thousandsof years (Gest and Mandelstam 1987; Cano and Borucki1995; Nicholson et al. 2000; Vreeland et al. 2000). In thedormant state, damage to crucial spore components (i.e.,DNA) accumulate and can only be repaired after the initia-tion of germination, the Wrst step in the return of spores tovegetative growth (Setlow and Setlow 1996; Setlow 2003,2006). It has been shown that the developmental cycle ofB. subtilis, consisting of postexponential-phase gene expres-sion, sporulation, dormancy, and germination, is controlledin large part at the transcriptional level (reviewed inPaidhungat and Setlow 2002; Piggot and Losick 2002;Setlow 2003).

The ability of microorganisms to survive and proliferatein their habitats depends on a variety of molecular mecha-nisms that adjust gene expression patterns in response tochanging environmental conditions (Bandow et al. 2002;Nicholson and Maughan 2002; Gaynor et al. 2005;Saint-Ruf and Matic 2006; Perkins and Nicholson 2008;Kang and Park 2010). DNA-dependent RNA polymerase(RNAP) is a central macromolecular machine controllingthe Xow of information from genotype to phenotype, andinsights into global transcriptional regulation can be gainedby studying mutational perturbations in the enzyme (Ing-ham and Furneaux 2000; Maughan et al. 2004; Perkins andNicholson 2008). RNAP is composed of an essential cata-lytic core enzyme (�2����) and one of several alternativesigma (�) factors (reviewed in Helmann and Moran Jr.2002). Because the RNAP holoenzyme contacts every pro-moter in the genome, single amino acid substitutions in

Communicated by Erko Stackebrandt.

R. Moeller (&) · G. ReitzResearch Group ‘Astrobiology’, Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center (DLR), Linder Hoehe, 51147 Cologne, Germanye-mail: ralf.moeller@dlr.de

I. VlaniTLaboratory of Evolutionary Genetics, Division of Molecular Biology, Ru²er BonkoviT Institute, Zagreb, Croatia

W. L. NicholsonSpace Life Sciences Laboratory, Department of Microbiology and Cell Science, University of Florida, Kennedy Space Center, FL, USA

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critical portions of the enzyme can lead to global changes ingene expression, hence in physiology and metabolism(Maughan et al. 2004; Perkins and Nicholson 2008).

Rifampicin (Rif) inhibits transcription initiation bybinding to the �-subunit of bacterial RNAP and blockingthe mRNA exit channel (Boor et al. 1995; Wehrli et al.1968; Wehrli and Staehelin 1971; Wehrli 1977; Campbellet al. 2001). Mutations in the nucleotide sequence withinthe rpoB gene encoding the �-subunit can cause rifampi-cin resistance (RifR). RifR mutations have been isolatedfrom numerous bacterial species and mapped to foursmall areas within the rpoB gene called Clusters I, II, III,and N (reviewed in Campbell et al. 2001; Severinov et al.1993). To date, all of the RifR mutations isolated inB. subtilis have been found to be single nucleotide substi-tutions resulting in speciWc amino acid changes within theN-cluster and Cluster I (Nicholson and Maughan 2002;Maughan et al. 2004; Perkins and Nicholson 2008). Previ-ous reports demonstrated that certain RifR mutationsaltering amino acids Q469, H482 and S487 in Cluster I ofB. subtilis rpoB exhibit alterations in fundamental physio-logical processes such as growth rate, competence fortransformation, sporulation, and germination (Rothsteinet al. 1976; Maughan et al. 2004; Perkins and Nicholson2008).

To date, however, limited information is availableregarding the impact of diVerent rpoB alleles on spore for-mation and spore resistance properties. Therefore in thiswork, we have examined the eVects of mutations in theB. subtilis rpoB gene on the regulation of sporulation initia-tion and spore resistance to obtain a better understanding ofthe mechanisms involved in multi-factorial nature of sporeresistance (as reviewed in Setlow 2006).

Materials and methods

Bacterial strains

Strains used in this work are listed in Table 1, and all arecongenic with the wild-type strain WN547 (MH5636). Inthis work, strain WN547 is referred to as the wild-typestrain with respect to RNAP, and all twelve congenicmutant strains are referred to by the amino acid substitutioncarried in their RpoB �-subunit.

Measurement of glucose catabolite repression during sporulation

To determine glucose catabolite repression during sporula-tion, the wild-type and the RifR rpoB mutant strains werecultured in liquid SchaeVer Sporulation Medium (SSM)(SchaeVer et al. 1965) with and without a supplement of1 % glucose under vigorous aeration (250 rpm) at 37 °C asdescribed previously (Maughan et al. 2006). After 24 h, thecolony-forming units (CFUs) before and after heat treat-ment (80 °C, 10 min) were determined to calculate thesporulation frequency (i.e., the ratio of heat-resistant spores(S) to total viable cells (V)) of the wild-type and the RifR

rpoB mutant strains as described in detail previously (Nich-olson and Setlow 1990; Maughan et al. 2006).

Spore preparation and puriWcation

Spores of all strains were obtained by cultivation under vig-orous aeration in SSM for 5 days at 37 °C until a sporula-tion frequency of >95 % had been reached, as judged byphase-contrast microscopy. Where appropriate, chloram-

Table 1 B. subtilis strains used in this study

Strain Genotype and/or phenotype Source (reference)

WN547 (MH5636)

trpC2 pheA1 cat 10His-rpoC; CmR Perkins and Nicholson (2008)

WN758 trpC2 pheA1 rpoB-Q469R cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN759 trpC2 pheA1 rpoB-H482R cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN760 trpC2 pheA1 rpoB-H482Y cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN761 trpC2 pheA1 rpoB-S487L cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN999 trpC2 pheA1 rpoB-S487F cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1000 trpC2 pheA1 rpoB-S487Y cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1002 trpC2 pheA1 rpoB-H482D cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1004 trpC2 pheA1 rpoB-V135F cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1007 trpC2 pheA1 rpoB-Q469K cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1009 trpC2 pheA1 rpoB-H482P cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1011 trpC2 pheA1 rpoB-Q469L cat 10His-rpoC; CmR RifR Perkins and Nicholson (2008)

WN1191a trpC2 pheA1 rpoB-A478D cat 10His-rpoC; CmR RifR This study

a The RifR rpoB allele A478D arose spontaneously from vege-tative cells of the B. subtilis strain 168 (Moeller et al. unpub-lished data) was transferred into strain WN547 and veriWed by PCR ampliWcation and nucleo-tide sequencing as described previously (15, 21), resulting in strain WN1191

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phenicol (Cm) (5 �g/ml) and/or Rif (50 �g/ml) were addedto the SSM. Sporulated cultures were harvested by centrifu-gation (10,000£g, 20 min, 4 °C) and treated with MgSO4

(2.5 �g/ml), lysozyme (200 �g/ml) and DNAse I (2 �g/ml)for 30 min at 37 °C in order to destroy the residual vegeta-tive cells. The enzymes were inactivated by heating for10 min at 80 °C. After repeated centrifugation and washingin distilled water, the puriWed spores (about 1010 spores/ml)were stored in aqueous suspension at 4 °C. Spore prepara-tions consisted of single spores with no detectable clumpsand were free (>99 %) of growing cells, germinated sporesand cell debris, as seen in the phase-contrast microscope(Nicholson and Setlow 1990; Moeller et al. 2007, 2011).

Measurement of spore resistance

To assay spore resistance to chemicals and heat, spore sam-ples consisting of 107 spores were exposed to heat (90 °Cwet heat or 100 °C dry heat) and chemicals (Wnal concen-trations of 2 % formaldehyde, 2 % glutaraldehyde, 5 %hydrogen peroxide in water) as previously described (Pop-ham et al. 1995; Riesenman and Nicholson 2000; Tennenet al. 2000; Paredes-Sabja et al. 2008). Spore recovery andviability assays were performed as described previously(Riesenman and Nicholson 2000; Tennen et al. 2000;Moeller et al. 2007, 2011).

Determination of spore viability and statistical analyses

The surviving fraction of B. subtilis spores was determinedfrom the ratio N/N0, with N the number of colony-formingunits (CFU) of the treated sample and N0 that of the non-treated controls. Spore inactivation curves, representing dose–eVect correlations, were obtained as described (Moeller et al.2011). Data are reported as LD90-values, the time (in h or min)required to kill 90 % of the initial spore population (Riesenmanand Nicholson 2000; Tennen et al. 2000; Moeller et al. 2007,2011). Data are expressed as averages § standard deviations.All experiments measuring spore resistance were repeated atleast three times, and data were compared statistically usingStudent’s t-test. Values were analyzed in multigroup pairwisecombinations, and diVerences with P values of ·0.05 wereconsidered statistically signiWcant (Riesenman and Nicholson2000; Tennen et al. 2000; Moeller et al. 2007, 2011).

Results

Glucose catabolite repression during spore formation in diVerent rpoB alleles

In B. subtilis, sporulation initiation is triggered when nutri-ent deprivation activates a protein phosphorylation cascade

(known as the “phosphorelay”) that ultimately phosphory-lates the Spo0A protein, the master transcriptional activatorof early sporulation genes (reviewed in Perego and Hoch,2003). Glucose-mediated catabolite repression prevents ini-tiation of sporulation by reducing the expression of geneswhose products are needed in the phosphorelay system(ShaWkhani et al. 2003a, b). Therefore, a standard assay tomeasure glucose repression of sporulation initiation is sim-ply to compare the sporulation eYciency of a strain culti-vated in SSM vs. SSM containing 1 % (v/v) glucose(SSM + G). To test the eVect of glucose catabolite repres-sion of sporulation initiation, we grew wild-type parentstrain WN547 and each of the 12 isogenic RifR strains at37 °C in liquid SSM and SSM + G, and the eYciency ofsporulation was quantiWed by determination of the ratio ofheat-resistant spores (S) to total viable cells (V) from 24-hcultures (Fig. 1). By 24 h all cultures had grown to equiva-lent cell concentrations, averaging between 2.91 £ 108 and8.25 £ 108 cfu/ml in SSM and SSM + G, respectively. Noculture deviated from the average by more than a factor of2.6, indicating that the mutant rpoB alleles exerted no dra-matic eVect on growth or 24-h cell yield. The wild-typeparental strain WN547 sporulated eYciently at 37 °C inSSM with (S/V = 0.35 § 0.12) and sporulation wasseverely repressed in SSM + G (S/V = 0.0045 § 0.0019)(Fig. 1), indicating the high eYciency of glucose acting assporulation repressor (Inaoka and Ochi 2007; Maughanet al. 2006; SchaeVer et al. 1965). Isogenic strains carryingthe Q469L, H482P, or S487Y mutations sporulated in SSMat higher eYciencies than did the parental strain, whereasstrains carrying the V135F, Q469K, Q469R, A478D,H482R, H482Y mutations sporulated at a signiWcantlylower frequency in SSM than the wild-type strain (Fig. 1).No signiWcant diVerences in the sporulation eYciency wereobtained in the strains carrying H482D, S487F and S487Lmutations (Fig. 1). In contrast, when analyzing sporulationeYciency in SSM + G, the strains carrying the Q469L,S487F, S487L, S487Y mutations sporulated at highereYciencies than the wild-type strain, whereas the straincarrying the H482Y mutation consistently sporulated at asigniWcantly lower frequency in SSM + G (Fig. 1). NosigniWcant diVerences in the sporulation eYciency inSSM + G were obtained for the strains carrying the V135F,Q469K, A478D, H482D, H482R mutations compared tothe wild-type rpoB strain (Fig. 1). The levels of glucosecatabolite repression during sporulation, as determined inthe ratio of the sporulation frequency in SSM with the spor-ulation frequency in SSM-G, were ranging from 203 to 1.1,with only the H482Y mutation causing a signiWcantlyhigher glucose catabolite repression rate compared with thewild-type rpoB strain (Fig. 2). Strains carrying the muta-tions Q469K, Q469L, A478D, H482P, H428R, S487F,S487L, and S487Y were all signiWcantly more glucose

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catabolite repression resistant than wild-type, whereasstrains carrying the V135F, Q469R, or H482D mutationsexhibited wild-type levels of catabolite repression of sporu-lation (Fig. 2).

Spore resistance to dry and wet heat

In order to determine the impact of altered rpoB alleles onthe spore resistance to heat, spores were prepared from thewild-type and the isogenic RifR strains were subjected to100 °C dry heat (Fig. 3a) and 90 °C wet heat (Fig. 3b).Semi-logarithmic plots of spore survival versus exposuretime yielded the time in h (dry heat) or min (wet heat)required to kill 90 % of the spore population (LD90), andthe average LD90 values from at least three separate experi-ments were compared (Fig. 3; Table 2). The LD90 value ofthe dry- and wet-heat-treated wild-type rpoB spores (with aHis-tagged rpoC) was 4.6 § 0.2 h and 17.4 § 3.5 min,respectively, which is in good agreement with previousobservations of B. subtilis spores with a normal RNAP(without a His-tagged rpoC) (reviewed in Nicholson et al.2000; Setlow 2006). Spores of strains carrying a V135F,Q469R, H482Y, and S487Y rpoB mutation were signiW-cantly more dry-heat-resistant than the wild-type, and onlythe spores of the Q469K rpoB mutant strain were signiW-cantly more dry-heat-sensitive (Fig. 3a). In all the othertested rpoB mutant spores, no signiWcant diVerences in thedry-heat resistance were observed (Fig. 3a). When compar-ing the LD90 values from the wet-heat spore treatments ofthe rpoB mutants, spores carrying the V135F, Q469L,Q469R, and H482P mutations were signiWcantly moreresistant to the wild-type spores, whereas spores carryingthe Q469K, A478D, H482D, S487L, or S487Y mutationswere signiWcantly more sensitive to wet heat than the wild-type spores (Fig. 3b). No signiWcant diVerences in wet-heatresistance were determined for spores carrying the H482R,H482Y, or S487F mutations (Fig. 3b). Interestingly, whentaken together, only spores carrying a V135F were signiW-cantly more resistant to both wet- and dry-heat exposure(Fig. 3). Of the 12 mutants tested, only spores carrying theH482R or S487F mutation showed wild-type spore resis-tance levels to both dry and wet heat (Fig. 3).

Spore resistance to chemicals

In addition to heat resistance, we measured the spore resis-tance to a variety of chemicals, namely 2 % formaldehyde,2 % glutaraldehyde, and 5 % H2O2 (Fig. 4), with these treat-ments chosen for comparison with literature data (asreviewed in Nicholson et al. 2000; Setlow 2006). The LD90

value of formaldehyde- and glutaraldehyde-treated wild-typerpoB spores (with a His-tagged rpoC) was 13.6 § 2.4 and23.6 § 1.8 min, respectively, and again those survival char-

Fig. 1 Glucose catabolite repression of sporulation in the wild-typeand Rifampicin-resistant (RifR) rpoB mutant strains of B. subtilis.Sporulation frequency (S/V) in the absence (white circles) and pres-ence (black circles) of 1 % glucose are presented. Data are expressedas averages § standard deviations (n = 3). Asterisks denote non-sig-niWcant diVerences (P > 0.05)

Fig. 2 Levels of glucose catabolite repression during sporulation inthe wild-type (white bar) and Rifampicin-resistant (RifR) rpoB mutantstrains (gray bars) of B. subtilis, as determined by the ratio of the spor-ulation frequency of the two diVerent sporulation approaches (with andwithout a glucose supplement). Data are expressed as averages andstandard deviations (n = 3). Asterisks indicate glucose cataboliterepression levels that were signiWcantly diVerent (P · 0.05) comparedto the wild-type rpoB strain

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acteristics were in good agreement with previous dataobtained from B. subtilis spores with a normal RNAP (with-out a His-tagged rpoC) (Loshon et al. 1999: Melly et al.2002; Paidhungat et al. 2000; Nicholson et al. 2000; Setlow2006). Spores of strains carrying the H482R, S487F, orS487Y mutations were signiWcantly more resistant to formal-dehyde, whereas spores of the V135F, Q469K, Q469R,A478D, or H482P mutations were more formaldehyde-sensi-tive than wild-type spores (Fig. 4a). No signiWcant diVer-ences in formaldehyde resistance were determined for sporescarrying the Q469L, H482D, or S487L mutations (Fig. 4a;Table 2). Determination of spore glutaraldehyde resistance(Fig. 4B) revealed a spectrum of resistance similar to that offormaldehyde resistance (Fig. 4a) for spores carrying the

V135F, Q469R, H482D, H482R, S487L, or S487Y muta-tions (Fig. 4b). DiVerences were noted in that: spores carry-ing the mutations Q469K and A478D were found to besigniWcantly more sensitive to formaldehyde but not glutaral-dehyde; spores carrying the Q469L and H482Y mutationswere signiWcantly more sensitive to glutaraldehyde but notformaldehyde; spores carrying the H482P allele were signiW-cantly more resistant to glutaraldehyde but not formaldehyde;and spores with the S487F mutation were more sensitive toglutaraldehyde but more resistant to formaldehyde than wild-type spores (compare Fig. 4a, b).

Analysis of resistance to 5 % hydrogen peroxide showedthat only spores carrying a S487L mutation were signiW-cantly more resistant to H2O2 than wild-type spores

Fig. 3 Resistance of spores from wild-type (black circle) and Rifampicin-resistant (RifR) rpoB mutant strains (white circles) of B. subtilis to 100 °C dry heat (a) and 90 °C wet heat (b). LD90 values are expressed as averages § standard deviations (n = 3). One asterisk indicates LD90 values that were signiW-cantly lower than wild-type spores, and two asterisks denote LD90 values that were signiW-cantly higher than wild-type spores (P · 0.05). Note the diVerence in the y-axes between a and b

Table 2 Survival characteristics of wild-type and rpoB mutant spores of B. subtilis

The characters (R, U, I) indicate reduced (R), increased (I) or unaVected (U) spore resistance compared to the wild-type rpoB strain

Data are averages and standard deviations (n = 3)a LD90 values (i.e., decimal reduction) in h (for the dry-heat treatment)b LD90 values (i.e., decimal reduction) in min (for wet heat, formaldehyde, glutaraldehyde, or hydrogen peroxide treatment)

RpoB genotype Dry heat (100 °C)a Wet heat (90 °C)b Formaldehyde (2 %)b Glutaraldehyde (2 %)b Hydrogen peroxide (5 %)b

Wild-type 4.6 § 0.2 17.4 § 3.5 13.6 § 2.4 23.6 § 1.8 43.7 § 1.9

V135F 6.5 § 0.4I 43.8 § 9.7I 6.0 § 1.2R 10.4 § 2.0R 32.9 § 2.6R

Q469K 5.9 § 0.3I 10.2 § 1.6R 1.4 § 0.3R 17.9 § 2.8R 29.7 § 2.9R

Q469L 4.9 § 0.4U 32.9 § 4.3I 15.9 § 3.6U 12.6 § 2.6R 29.1 § 2.3R

Q469R 3.9 § 0.1R 30.8 § 5.4I 3.6 § 0.7R 9.0 § 3.2R 40.5 § 2.8U

A478D 4.5 § 0.3U 8.4 § 2.8R 4.8 § 0.6R 16.7 § 4.4R 28.7 § 3.9R

H482D 5.1 § 0.4U 5.7 § 0.4R 13.4 § 2.2U 21.7 § 3.4U 28.4 § 2.6R

H482P 4.5 § 0.3U 72.6 § 8.1I 8.5 § 3.1R 30.1 § 2.1I 46.5 § 1.3U

H482R 4.8 § 0.4U 12.8 § 2.1R 24.9 § 2.7I 28.7 § 2.4I 40.1 § 3.8U

H482Y 6.3 § 0.5I 13.3 § 2.7R 14.9 § 2.4U 16.5 § 2.9R 33.1 § 1.9R

S487F 4.7 § 0.3U 18.7 § 4.8U 20.2 § 4.1I 11.9 § 2.4U 34.2 § 1.4R

S487L 5.1 § 0.5U 8.8 § 1.2R 12.6 § 2.7U 24.2 § 3.8U 51.4 § 3.1I

S487Y 6.5 § 0.4I 8.2 § 0.6R 26.9 § 3.3I 32.7 § 2.6I 46.3 § 3.4U

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(Fig. 4c). Spores of strains carrying the Q469R, H482P,H482R, or S487Y behaved essentially like wild-type spores(Fig. 4c), while spores carrying a V135F, Q469K, Q469L,A478D, H482D, H482Y, or S487F exhibited signiWcantlower H2O2 resistance levels than the wild-type rpoBspores (Fig. 4c). Of all the rpoBi mutants tested, only thestrain carrying the V135F mutation was signiWcantly moresensitive to all chemical treatments than were wild-typespores (Fig. 4; Table 2).

Discussion

It has been shown that the developmental cycle of B. sub-tilis, consisting of postexponential-phase gene expression,sporulation, dormancy, and germination, is controlled inlarge part at the transcriptional level (reviewed in Paidhun-gat and Setlow 2002; Piggot and Losick 2002). Here, wepresent data indicating that alterations in closely linkedamino acids residing within rpoB N-cluster (V135F) andcluster I (Q469, A478, H482, and S487) not only conferRifR, but also exert a range of eVects on the cataboliterepression of sporulation initiation and on spore resistanceproperties. In addition to the RifR phenotype, each aminoacid change in rpoB resulted in its own unique spectrum ofcatabolite repression and spore resistance (Fig. 1; Table 2).

Glucose catabolite repression

Cells enter the pathway to sporulate in response to conditionsof nutrient limitation, which results in the formation of thepredivisional cell (Wang et al. 2006). The initiation of sporu-lation is a complex process by which environmental signalsare sensed by the cell and transduced via a phosphorelay sys-tem to the master transcriptional regulator Spo0A (reviewedin Perego and Hoch 2002). Glucose is one of the most eVec-tive repressors of sporulation initiation (SchaeVer 1969), andits eVect has been traced to down-regulation of transcriptionof genes encoding components of the phosphorelay system(spo0F, kinA) and to spo0A itself (ShaWkhani et al. 2003a, b).A catabolite-resistant sporulation (Crs) phenotype was previ-ously found to result from a missense mutation (crsA47;leading to P290F) in the sigA (rpoD) gene encoding the

Fig. 4 Resistance of spores from wild-type (black circle) and Rifam-picin-resistant (RifR) rpoB mutant strains (white circles) of B. subtilisto 2 % formaldehyde (a), 2 % glutaraldehyde (b), and 5 % hydrogenperoxide (c). LD90 values are expressed as averages § standard devia-tions (n = 3) as described in the text. One asterisk indicates LD90 val-ues that were signiWcantly lower than wild-type spores, and twoasterisks denote LD90 values that were signiWcantly higher thanwild-type spores (P · 0.05). Note the diVerence in the y-axes betweena, b and c

�

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major vegetative RNAP sigma-A (�A) factor (Kawamuraet al. 1985). Thus, the direct involvement of the vegetative�A subunit of RNAP in glucose catabolite repression of spor-ulation has been demonstrated. In our experiments, strainscarrying the Q469L, S487F, S487L, S487Y mutations inrpoB also exhibited a strong Crs phenotype (Fig. 1), thus alsoimplicating the �-subunit of RNAP in glucose cataboliterepression of sporulation initiation. It is interesting to notethat in a separate but relevant study, Nanamiya et al. (2000)isolated a mutation in sigA (E314K) that caused sporulationinitiation to be temperature sensitive. A second-site repressormutation restoring sporulation at high temperature was iso-lated and subsequently mapped also to the rpoB gene(A863G), strongly implicating direct interaction between the� and � subunits of RNAP in both activation and cataboliterepression of sporulation initiation (Nanamiya et al. 2000;Helmann and Moran Jr. 2002).

Spore resistance properties

Spores are extremely resistant to being killed by heat and avariety of cytotoxic chemicals, and spore resistance propertiescan be traced to unique structures of the spore, such as a highlycross-linked protein coat, a modiWed peptidoglycan spore cor-tex, a low core water content and abundant intracellular sporecore constituents such as the calcium chelate of dipicolinic acid(Ca-DPA) and �/�-type small, acid-soluble spore proteins(�/�-type SASP), the latter two of which protect spore DNA(Hackett and Setlow 1988; Popham et al. 1995; Driks 1999;Nicholson et al. 2000; Paidhungat et al. 2000; Setlow et al.2000; Lee et al. 2008). The genes responsible for these struc-tural proteins and spore-speciWc enzymes are transcriptionallycontrolled in either the developing forespore or mother cellduring diVerentiation (for extensive reviews, see Piggot andLosick 2002; Driks 1999, 2002; Paidhungat and Setlow 2002),thus alterations in RNAP aVecting promoter binding and selec-tivity might be predicted to result in altered spore properties. Inmost (but not all) cases, RifR mutations in rpoB resulted inlowering of spore resistance to various chemical treatments,but complete concordance was only seen in the V135F muta-tion, which signiWcantly lowered spore resistance to formalde-hyde, glutaraldehyde, and H2O2 (Fig. 4).

Although speciWc rpoB mutations appeared to increaseor decrease the chemical, wet-heat, or dry-heat resistance ofspores (Figs. 3, 4), no clear-cut pattern could be discerned;it thus appears that each RifR mutation exerted its own spe-ciWc spectrum of eVects on each property tested, summa-rized below.

V135F

The V135F strain showed no diVerences in the glucosecatabolite repression during spore formation (Fig. 1). Inter-

estingly, V135F spores were more resistant to heat, butmore sensitive to the tested chemicals than wild-type rpoBspores (Figs. 3, 4).

Q469

We tested three diVerent Q469 alleles (K, L, R) on theircatabolite repression and spore resistance. Surprisingly, thestrain carrying a Q469L mutation was resistant to cataboliterepression of sporulation initiation, while the Q469K andQ469R alleles were not (Fig. 2). In regard to their spore heatresistance, only Q469K spores were more resistant to dryheat than wild-type spores, whereas the Q469L and Q469Ralleles were signiWcantly more resistant to wet heat thanwild-type spores (Fig. 3). None of the Q469 alleles was moreresistant to the three tested chemicals than the wild-type(Fig. 4); in fact, spores of the Q469K mutant were most sen-sitive to formaldehyde of all strains tested (Fig. 4a).

A478D

Sporulation of the A478D strain was substantially more glu-cose repression-resistant than the wild-type strain (Fig. 2).A478D spores showed the same level of resistance to dryheat than the wild-type, but a weaker level in spore resistanceto wet heat and the three tested chemicals (Table 2).

H482

At codon H482, we were able to compare four diVerentamino acid substitutions (D, P, R, and Y), all previouslyisolated as spontaneous mutations in both vegetative cellsand spores (Nicholson and Maughan 2002; Perkins et al.2008). Only the H482R mutant was found to be resistant toglucose catabolite repression (Fig. 2). Only spores of theH482Y strain were more dry-heat resistant than wild-typespores, while the H482D and H482P strains showed signiW-cantly lower or higher wet-heat resistance, respectively(Fig. 3). Regarding spore resistance to chemicals, the H482mutants exhibited a variety of patterns (Fig. 4). Interest-ingly, spores of the H482R were more resistant than wild-type spores to formaldehyde and glutaraldehyde, but not tohydrogen peroxide, wet or dry heat.

S487

With codon 487 of rpoB, we had the opportunity to com-pare the physiological eVects of replacing the polar aminoacid S with either a non-polar (L) or an aromatic (F, Y)amino acid. Considering the rather dramatic chemicaldiVerences, the amino acid changes would be predicted toconfer, all three mutants exhibited glucose catabolite repres-sion resistance (Fig. 1), whereas numerous allele-speciWc

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766 Arch Microbiol (2012) 194:759–767

diVerences in spore resistance to heat and chemicals wereobtained (Figs. 3, 4; Table 2).

Conclusions

Our results lend support to the hypothesis that the �-subunitof RNAP plays a much wider regulatory role in the sporeformation under glucose-limiting and -abundant conditionsas well as in the spore resistance to heat and cytotoxicchemicals than has previously been appreciated. Recently,Keijser et al. (2007) identiWed several key transcriptionalevents during the Wrst 2 h of spore germination, showingdirect correlations between transcriptional and cellularactivity. It seems apparent that a well-developed regulatorymechanism exists for the integration of key physiologicalevents during germination and outgrowth. They suggestedthat there are checkpoint processes interacting on DNAintegrity, metabolic status, the initiation and prolongationof spore germination toward vegetative growth (Wang et al.2006; Keijser et al. 2007). Previous reports by Maughanet al. (Maughan et al. 2004) and others (e.g., Nanamiyaet al. 2000; Perkins and Nicholson 2008) showed that RifR

rpoB mutations deWnitively eVect the regulation of devel-opmental processes during sporulation and germination byboth locus-speciWc and allele-speciWc eVects. However, theresults reported here clearly establish the importance of theRNAP in the initiation of sporulation as well as in the sporeresistance to heat and chemicals. At present, the exactmechanism(s) involved in regulatory role of RNAP isobscure, but it is certainly amenable to experimental eluci-dation. We are directing current experiments toward a deW-nition of how the diVerent rpoB alleles aVect in vivoexpression of the complete B. subtilis transcriptome duringsporulation and spore germination (with and without priorexposure to damaging agents or physical stressors). Wepredict that such studies will lead to the identiWcation ofkey developmental genes whose expression is altered inrpoB mutants and which are thus dependent upon the regu-latory function of the RNAP �-subunit.

Acknowledgments The authors are very grateful to Andrea Schrö-der and Amy Perkins for their skillful technical assistance during partsof this work. We express thanks to Krunoslav BrbiT-KostiT for hisvaluable support and the three anonymous reviewers for their construc-tive comments and insightful suggestions. This study was supported inpart by grant CBP.EAP.CLG.983747 from the NATO Science forPeace Program to R.M., and by grants from NASA (NNA06CB58Gand NNX08AO15G) to W.L.N.

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